This invention is directed to test devices for determination of characteristics of samples, unitized housings, and kits incorporating the test strips and housings, and methods of determining the characteristics of samples using the test strips and housings.
Among the many analytical systems used for detection and/or determination of analytes, particularly analytes of biological interest, are chromatographic assay systems. Among the analytes frequently assayed with such systems are: (1) hormones, such as human chorionic gonadotropin (hCG), frequently assayed as a marker of human pregnancy: (2) antigens, particularly antigens specific to bacterial, viral, and protozoan pathogens, such as Streptococcus, hepatitis virus, Giardia, and feline leukemia virus (FeLV); (3) antibodies, particularly antibodies induced as a result of infection with pathogens, such as antibody to the bacterium Helicobacter pylori, to human immunodeficiency virus (HIV), or to feline immunodeficiency virus (FIV); (4) other proteins, such as hemoglobin, frequently assayed in determinations of fecal occult blood, an early indicator of gastrointestinal disorders such as colon cancer; (5) enzymes, such as aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, and glutamate dehydrogenase, frequently assayed as indicators of physiological function and tissue damage; (6) drugs, both therapeutic drugs such as antibiotics, tranquilizers, and anticonvulsants, and illegal drugs of abuse, such as cocaine, heroin, and marijuana; (7) environmental pollutants such as pesticides and aromatic hydrocarbons; and (8) vitamins.
Such chromatographic systems are frequently used by physicians, veterinarians, and medical technicians for rapid in-office diagnosis and therapeutic monitoring of a variety of conditions and disorders. They are also increasingly used by patients and animal owners themselves for at-home monitoring of such conditions and disorders.
Among the most important of such systems are the "thin layer" systems in which a solvent moves across a thin, flat, absorbent medium. Among the most important of tests that can be performed with such thin layer systems are immunoassays, which depend on the specific interaction between an antigen or hapten and a corresponding antibody. The use of immunoassays as a means of testing for the presence and/or amount of clinically important molecules has been known for some time. As early as 1956, J. M. Singer reported the use of an immune-based latex agglutination test for detecting a factor associated with rheumatoid arthritis (Singer et al., Am.J. Med. 22:888-892 (1956)).
Among the chromatographic techniques used in conjunction with immunoassays is a procedure known as immunochromatography. In general, this technique uses a disclosing reagent or particle that has been linked to an antibody to the molecule to be assayed, forming a conjugate. This conjugate is then mixed with a specimen, and if the molecule to be assayed is present in the specimen, the disclosing reagent-linked antibodies bind to the molecule to be assayed, thereby giving an indication that the molecule to be assayed is present. The disclosing reagent or particle can be identifiable by color, magnetic properties, radioactivity, emission of light, specific reactivity with another molecule, or another physical or chemical property. The specific reactions that are employed vary with the nature of the molecule being assayed and the sample to be tested.
Immunochromatographic assays fall into two principal categories: "sandwich" and "competitive," according to the nature of the antigen-antibody complex to be detected and the sequence of reactions required to produce that complex. The antigen to be detected can itself be an antibody, such as in serological assays for H. pylori-specific antibody or for antibody to FIV. In such cases, the antibody to be detected can be bound to a specific antigen. Alternatively, the antigen to be detected can be detected indirectly by using a labeled second antibody that binds the first antibody to the analyte to be detected.
In general, the sandwich immunochromatographic procedures call for mixing the sample that may contain the analyte to be assayed with antibodies to the analyte. The antibodies are mobile and typically are linked to a label or a disclosing agent, such as dyed latex, a colloidal metal sol, or a radioisotope. This mixture is then applied to a chromatographic medium containing a band or zone of immobilized antibody to the analyte of interest. The chromatographic medium is often in a form of a strip resembling a dipstick. When the complex of the molecule to be assayed and the labeled antibody reaches the zone of the immobilized antibodies on the chromatographic medium, binding occurs, and the bound labeled antibodies are localized at the zone. This indicates the presence of the molecule to be assayed. This technique can be used to obtain quantitative or semi-quantitative results.
Examples of sandwich immunoassays performed on test strips are described by U.S. Pat. No. 4,168,146 to Grubb et al. and U.S. Pat. No. 4,366,241 to Tom et al., both of which are incorporated herein by this reference.
In competitive immunoassays, the label is typically a labeled analyte or analyte analog which competes for binding of an antibody with any unlabeled analyte present in the sample. Competitive immunoassays are typically used for detection of analytes such as haptens, each hapten being monovalent and capable of binding only one antibody molecule. Examples of competitive immunoassay devices are those disclosed by U.S. Pat. No. 4,235,601 to Deutsch et al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No. 5,208,535 to Buechler et al., all of which are incorporated herein by this reference.
Although useful, currently available chromatographic techniques using test strips have a number of drawbacks. Many samples, such as fecal samples, contain particulate matter that can clog the pores of the chromatographic medium, greatly hindering the immunochromatographic process. Other samples, such as blood, contain cells and colored components that make it difficult to read the test. Even if the sample does not create interference, it is frequently difficult with existing chromatograph test devices to apply the sample uniformly to the chromatographic medium. This is highly desirable to ensure that the sample front moves uniformly through the chromatographic medium to insure that the sample reaches the area where binding is to occur in a uniform, straight-line matter. Other problems exist with currently-available test strips because of the nature of the sample to be assayed or the assay to be carried out. In many currently-available test strips, the time of passage of the specimen, from the point of application to passage past the specific capture band on the solid phase, frequently results in an undesirable time delay in obtaining results. In addition, variable specimen and reagents may be lost in the dead volume of the elements in the path to the capture zone.
With currently-available designs, it is also impractical to perform washing steps which are frequently desirable to improve sensitivity and reduce background. Also, it is difficult, and in many cases impossible, to carry out preincubation steps within the device.
Sample preparation and waste generation are responsible for other problems with currently available devices and techniques for immunochromatography. The increased prevalence of diseases spread by infected blood and blood fractions, such as AIDS and hepatitis, has exacerbated these problems. It is rarely possible to apply a sample (such as feces) or a sampling device (such as a throat swab) directly to the chromatographic medium. Several extraction and pretreatment reactions are usually required before the sample can be applied to the chromatographic medium. These reactions are typically carried out by the physician, veterinarian, or technicians performing the test in several small vessels, such as test tubes or microfuge tubes requiring the use of transfer devices such as pipettes. Each of these devices is then contaminated and must be disposed of using special precautions so that workers or people who may inadvertently come into contact with the waste do not become contaminated.
Still another limitation in chromatographic devices currently available for use by the physician, veterinarian, or technician is their inability to perform two-directional or two-dimensional chromatography. These techniques have long been known to be powerful analytical tools, but their complexity relative to simple unidirectional chromatography has made it difficult to apply them to test strip devices in the physician's office or a clinical laboratory.
Additionally, currently available test devices cannot perform two independent assays for two different analytes on the same test strip. One particular application of this would be the ability to perform a unidirectional sandwich assay and a bidirectional serological assay for an antibody as an analyte in the same test strip. Because the antibody that binds to a particular antigen is only a small fraction of the total antibody molecules present in the serum, the use of a unidirectional assay for an analyte that is an antibody is generally unsatisfactory, because the detection reagent will bind to many other antibody molecules on the test strip other than the antibody for the desired antigen, thus creating an unacceptably high background. This is true even if a second antibody specific for a class or subclass is used, because many individual antibodies belong to that class or subclass.
The ability to perform two such immunoassays on the single test strip is desirable when it is desired to determine the existence or non-existence of two specific diseases or conditions in the same sample. Alternatively, it can be desirable to perform assays for the simultaneous detection of both an antigen that is associated with a viral or bacterial pathogen and an antibody that is associated with the immunological response of the body to that pathogen in the same sample. An example is HIV virus, where a protein antigen known as p24 can be found in infected patients, while an antibody to the virus can also be found in many patients. It can be desirable to assay both of these in order to help determine the clinical status of the patient.
Accordingly, there is a need for an improved assay device capable of handling a broad range of chromatographic assays, including the ability to assay for two separate analytes in the same test strip. Such a device should be capable of receiving a possibly contaminated sample or a sample preparation device directly so as to eliminate the need for extraction vessels and transfer devices. Such a device, preferably in the form of a test strip, should also be capable of performing immunochromatographic assays on colored samples or samples containing particulates without interference and should be able to deliver the sample to the chromatographic medium uniformly and evenly to improve accuracy and precision of the tests. Moveover, such a test strip should minimize the time delay experienced in the performance of the assay and also minimize the dead volume in order to provide maximum economy in the use of samples and reagents.